Supplement to R21: Mechanism and in vivo activity of novel glycan-based therapy against flavivirus endothelial permeability and vascular leak Evaluation of therapeutics targeting SARS-CoV-2 infection and defining pathogenic mechanisms of SARS-CoV-2-triggered pulmonary dysfunction Abstract The emerging severe acute respiratory syndrome-corona virus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is spreading rapidly across the world, already affecting 199 countries, and predicted to infect up to 60% of the population, with a ~4.4% case fatality rate to date. Novel therapeutics are desperately needed, and as we are currently investigating anti-flavivirus properties of cyclodextrin compounds (CDs), which have broad antiviral activity, we propose here to expand this investigation to test these compounds for anti-SARS-CoV-2 activity in human bronchial epithelial cells (Aim 1). First, we will fast-track the FDA-approved CDs in our collection, and then screen the rest. OSC Dr. Ralph Baric (UNC) will confirm our most promising candidates in primary human cells and collaborate on setting up further studies to test them in his mouse models. Based on previous literature and data acquired through the parent grant, we hypothesize that these compounds may have direct virucidal activity by inactivating virions, as well as potentially inhibiting cell attachment by blocking the SARS-CoV-2 spike glycoprotein (S) from interacting with glycans and/or the viral receptor angiotensin converting enzyme 2 (ACE2) on the cell surface. Though infected patients succumb to acute respiratory distress syndrome (ARDS) involving vascular leak, the viral triggers of this pathology are unclear. Experiments with SARS-CoV-1 found that internalization of ACE2 along with virus particles upon infection reduces ACE2 levels on the cell surface, resulting in increased angiotensin II activity. The angiotensin II activity is believed to result in upregulation of vasoactive molecules such as vascular endothelial growth factor F (VEGF) and disruption of intercellular junctions, both inducing vascular leak. In Aim 2, our optimized system for the study of endothelial cell dysfunction, resulting from many years of work with flaviviruses and included in the parent grant, will be applied to investigate SARS-CoV-2 vascular pathology induced by the viral S protein and secondary mediators like VEGF. As we have already observed in vitro anti-leak as well as antiviral properties for some of the tested CDs, we also propose to test these candidates as therapeutics to treat COVID-19 disease manifestations. As such, this supplemental grant proposal has the potential to define triggers of SARS-CoV-2 S- mediated vascular leak, contributing to ARDS, as well as testing CDs as therapeutics targeting viral infection directly and indirectly via downstream pathogenesis. FDA-approved CDs and derivatives that prove to be effective as COVID-19 treatments have the potential to be rapidly developed for potential use in patients. 1